Process for preparing l-lysine

ABSTRACT

L-Lysine is prepared by biochemical asymmetrical hydrolysis of Alpha -amino- Epsilon -caprolactam.

United States Patent Fukumura 1 Nov. 6, 1973 [54] PROCESS FOR PREPARINGL-LYSINE [58] Field of Search 195/29 [75] Inventor: Takashi Fukumura,Kamakura,

Japan [56] References Cited [73] Assignee: Toray Industries, Inc.,Tokyo, Japan UNITED STATES PATENTS 3,056,729 10/1962 Seto 195/29 [22]Flled: Nov. 10, 1971 [21] Appl. No.: 197,487 Primary Examiner-Alvin E.Tanenholtz Attorney-Austin R. Miller et a1.

[30] Foreign Application Priority Data [57] ABSTRACT Nov. 19, 1970 Japan45/101472 y is p p y biochemical asymmetrical y 52 us. Cl. 195/29 dmlyssInt. Cl C12d 13/06 4 Claims, No Drawings l I PROCESS FOR PREPARINGL-LYSINE DESCRIPTION OF THE PRIOR ART L-Lysine is a wellknown essentialamino acid. It is known that L-lysine is produced by hydrolysis ofa-amino-e-caprolactam. (I-Ielv. Chim. Acta, XLI, P 186 [1958]).

In respect to microbiological means a process is described by Seto etal. in US. Pat. No. 3,056,729. But the rate of conversion ofDL-a-amino-e-caprolactam to L-lysine in the process is at most 30percent.

SUMMARY OF THE INVENTION This invention relates to a new and usefulmethod for preparing L-lysine quantitatively from L-a-amino-ecaprolactamin substantially pure form, i.e., free of any contamination with theD-lysine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with thisinvention, the hydrolyzing activity of the microorganisms belonging toCryptococcus, Candida, Trichosporon and the enzyme produced by saidmicroorganisms brings about the conversion of L-a-amino-e-caprolactam toL-lysine via a selective hydrolytic step, whereby the desired product isreadily obtained in substantially pure form and quantitatively. In otherwords, in accordance with the process of this invention,L-aamino-e-caprolactam gives pure L-lysine and DL-a-amino-e-caprolactamgives 2 pure ll -lysine and D-a-arnino-ecaprolactam. (Hereinaftera-amino-e-caprolactam is referred to as aminolactam).

It has been found that L-amino-lactam is converted 5 to L-lysine whenincubated in contact with a cell suspension of any one of several yeaststrains isolated from soil. Subsequently, several strains ofCryptococcus laurentii, Candida humicola and Trichosporon cutaneum fromIFO were also found to possess the L-aminol0 lactam hydrolyzingactivity.

The L-amino-lactam hydrolyzing activity was also manifested in a growingculture, a culture broth, treated (or artificially modified) cells, suchas vacuumdried cells, lyophilized cells and acetone-dried cells, a

cell extract, and an enzyme, namely L-amino-lactam amidohydrolase.

The yeast strain which is employed in the present invention can bechosen from the strains of the genera to which the yeast samples giventhe PERM-P. 709, 710,

712, 714, 715, 717, 719,725,1FO 0753 (ATCC 9949) and [F0 0760, 0173,1198, 0609, 0906 are taxonomically belong. (PERM-P is a Deposit numberin the Fermentation Research Institute in Japan.)

According to our classification, strains of PERM-P 709 and 710 werefound to be Cryptacoccus laurentii, strains of PERM-P 715, 717 and 719were Candida humicola, and strains of FERM-P 712, 714 and 725 wereTrichosporan cutaneum.

In Table I characteristics of the FERM-P 709, 710

and Cryptococcus laurentii IFO 0609, 0906 are presented.

TABLE 1 g A H g 7 HM V V ERA 13159; I E Y Cryptococcus CryptococcusCryptococciis cryptococcus laurentii laurentii laurentii laurentiiSpecific name IFO 0609 IFO 0906 TORAY 2001 TQRAY 2002 1. Microscopicobservations (1) Cells in YM broth I 1. Shape M round, oval, long oval,elongate round, oval, long oval 2. Dominant cells oval, (4-5) X (8-9) ,around to oval, (2.5-4) X (3-5) a 3. Vegetative reproduction.

multilateral budding (2) Slide culture on potatoagar.

(3) Ascospore formation not observed on a gypsum block or a modifiedGorodkowa-agar in 30 days incubation.

11. Cultural characteristics (1) YM broth 1. Turbidity not turbid 2.Sediment compact 4. Pellicle creeping occasionally creeping 4. Ringabsent occasionally very weak ring (2) YM-agar colonies 1. Shapecircular 2. Margin entire COIIVCX 4. Surface smooth, dull smooth,glistening 5. Quality butyrous mucous 6. Color light reddish yellowlight cream-colored I11. Physiologicalcharacteristics (1) Sugarfermention no gas produced from glucose, galactose. sucrose, maltose,lactose, and

raffinose m 30 days incubation.

" TABLE l-Continued FERM P709. FERM P710. Cryptococcus Cryptococcus(ryptocoucus (ryptococcus laurentii lnurentii lziurentii luurcntiiSpecific name LIFO 060) IFO 0906 TURAY 1001 TOR/\Y 2002 (2) Sugarassimilation (auxanographic method) '(3) KNO assimilation ilated.

(4) Litmus milk 1. Color 2. Coagulation 3. Peptonization no (5)Splitting of arbutin glucose. galnctose. sucrose. maltose-iltiet oseundmn'infitlir55am absent (both on liquid culture and auxanographic method)slightly acid, after 29 days reduced coagulated after 26 days positive(strong) positive (violet reaction by lugol positive (blue reaction bylugol solution) (7) L-Lysine utilization utilized as carbon and (8)l.-/\minolactam utilizautilized as carbon and tion (9) Growth conditionsIV. Source nitrogen sources nitrogen sources good growth at 30 C goodgrowth at pH 6.0

isolated from soil seliblsirceiJcrypzaeaeas 15mm; $66609, IFO 0906.

In Table 2 characteristics of the PERM-P 715, 717,

30 719 and Candida humicola IFO 0753 (=ATCC 9949) are presented.

' TABLE 2 v i 7 FETYVFPfiI. FERM- P115. FE IZ Candida Candida candldaCandida humicola humicola humicola humicola Specific name IFO 0753 Toray2010 'Ijoray 2008 W Toray 2020 I. Microscopic observations (1) Cells inYM broth 1. Shape variable shape: round, lemon-shaped, oval, elliptical,elongate, cylindrical.

2. Dominant cells round to oval, elliptical, (4-5) X (7-10) round tooval,

3. Vegetative reproduction.

(2) Slide culture on potatomultilateral budding (3) Ascospore formation11. Cultural characteristics (1) YM broth not observed on a gypsum blockor a modified Gorodkowa-agar in 30 days incubation.

l. Turbidity not turbid 2. Sediment compact flocculent 3. Pellicleislets to thin pellicle no formation 4. Ring clear ring obscure ring (2)YM-agar colonies 1. Shape circular, becoming filamentous 2. Marginentire, becoming filamentous TABLE 2.Continued ifi'ccfs ii EER -Elli.PERM-P719. Candida Candida Candida Candida humicola humicola humicolahumicola Specific name IFO 0753 Toray 2010 Toray 2008 Toray 2020 carer.c';,;;a"" Starr as.

times umbonate umbonate umbonate 4. Quality butyrous, latter mucoidbutyrous 5. Color yellowish light cream-colored (3) YM slant culture 1.Growth abundant moderate abundant 2. Margin filamentous entire undulatefilamentous 3. Surface wrinkled. smooth. dull rough. dull rough andlusterless wrinkled. dull III.

ITis satisfactory to consider the three strains of FERM-P 715, 717, 719asCandida and more particularly Candida humicola on the basis of thedescription in J. Lodder and N. J. W. Kreger Van Rijs The 45Physiological characteristics (1) Sugar fermentation (2) Sugarassimilation (auxanographic method) (3) KNOa assimilation no gasproduced from glucose. galactose. sucrose, maltose. lactose andraffinose in 30 days incubation.

glucose, galactose, sucrose. maltose. lactose are assimilated absent(both on liquid culture and auxanographic methods).

(4) Litmus milk 1. Color slightly red, later reduced unchanged. after 22days reduced 2. Coagulation coagulated coagulated after 25 days 3.Peptonization after 30 days slow peptonization of slow peptonization ofthe coagulathe coagulation.

tion.

(5) Splitting of arbutin (6) Starch-like compound (7) L-Lysineutilization (8) L.Aminolactam utiliza' tion.

(9) Growth conditions Source isolated from soil Yeastsj fl TEXEn omiESEuHy X 1967) and the close resemblance to Candida humicola IFO 0753.

In Table 3, characteristics of the'FERM-P 712, 714, 725 and Trichosporoncutaneum IFO 0173 are presented.

TABLE 3 u wig ji tiTi/ljlfjz FERfM' FI'fi, 'FET/I FTIEI Trichosporon Trichosporoh Tricliosporon Trichosporon cutaneum cutaneum cutaneum cutaneumSpecific name [F0 0173 Toray 2007 Toray 2035 Toray 2004 1. Microscopicobservations (1) Cells in YM broth 1. Shape variable shape: round, oval,long oval, cylindrical, occasionally giant 2. Dominant cells 3.Vegetative reproduction multilateral budding and fission mainly fission(2) Slide culture and potatoagar cells present. rich development of truemycelium. no pseudomycelium.

blastospores round to oval forming clusters. arthrospores in typicalzigzag form.

('3) Ascospore formation not observed on gypsum block or a modifiedGorodkowa-agar in 30 days incubation.

II. Cultural @qacteristics (l) Wbroth l. Turbidity not turbid TABLE3.Continued V. 7-. EEEM:E7H1.. FEFMFPZZL EEKW m w Tiichos poronTrichosporon Trichosporon Trichosporon cutaneum cutaneum cutaneumcutaneum 7 Specific name [F0 0173 Toray 2007 Toray 2035 Toray 2004 ""2."Sediment compact ricuiem W m g 3. Pellicle pellicle formed, later goingdown as sediment (2) YM-agar colonies 1. Shape circular. becomingfilamentous 2. Margin filamentous entire, becoming filamentousfilamentous 3. Elevation raised convex convex. later raised umbilicate'smooth. 4. Surface rough smbooth. I flbbqmmg wrinkled gg: velvet-likeiii dull i V jgi gg lusterless 5. Quality butyrous to dry butyrous dry6. Color cream-colored cream to whitish cream colored (3) YM slantculture LGrowth abundant moderate abundant 2. Margin filamentousundulate. undulate to filamentous becoming filamentous on the upper part3. Surface (30 days culvelvet-like rough velvet-like wrinkled ture)folded folded Ill. Physiologicalcharacteristics (1) Sugar fermentation Yrafi'rnose in 30 days incubation no gas produced from glucose.galactose, sucrose. maltose. lactose.

(2) Sugar assimilation (auxar iographic metbndt (3) KNO assimilationglucose. galactose. sucrose. maltose. lactose are assimilated absent(both on liquid culture and auxanographic methods) (4) Litmus milk 1.Color unchanged. after days reduced 2. Coagulation coagulated after 25days (6) Starch-like compound not produced (7) L-Lysine utilizationutilized as carbon and nitrogen sources (8) lllminolaaam utilizationutilized as carbon and nitrogen sources (9) Growth conditions goodgrowth at C good growth at pH 6.0

IV. Source isolated from soil 7 It is satisfactory to consider the threestrains of FERM-P 712, 714, 725 as Trichosporon and more particularlyTrichospororr curaneum on the basis of the description in J. Lodder andN. J. W. Kreger Van Rijs The Yeasts, A Toxonomic Study (I967) and theclose resemblance to Trichosporon curaneum IFO 0173.

As for the composition of the culture medium, either a synthetic or anatural culture medium is suitable so long as it contains the essentialnutrients for the growth of the yeast strain employed, and a smallamount of L- or DL-amino-lactam as the inducer for the enzyme. Forinstance, the medium employed in the examples of this invention consistof 1% glucose, 0.3% NH,N0,, 5% L- amino-lactam hydrochloride, 0.1% KHJO0.05%-

MgSO,'7H O, 0.02% MnCl SH O and 0.05 percent yeast extract (pH 6).

Cultures are carried out aerobically at 2040 C, preferably 30 C.

As for the state of the L-amino-lactam hydrolyzing agent when applied inthe process of this invention, growing cells, a culture broth, livingcells, modified cells such as lyophilized cells and acetone-dried cells,a cell extract, and a purified protein derived from cells, namelyL-amino-lactam amidohydrolase" are effectively used. Moreover, theenzyme can be also used in the state of an insolubilized enzyme such asDEAE-, TEAE-, GE-cellulose-adsorbed enzyme and DEAE- sephadex-adsorbedenzyme. Furthermore, as for the L-amino-lactam hydrolyzing agent, theL-aminolactam amidohydrolase the characteristics of which are describedin the next paragraph is specified. Therefore, microorganisms other thanCryptococcus, Candida and Trichosporon are also employed as producers ofthe L- amino-lactam hydrolyzing agent so far as they produce the sameL-amino-lactam amidohydrolase as specified in the present invention.

The characteristics of the L-amino-lactam amidohydrolase are asfollows: 1. Catalytic action: hydrolyze L-amino-lactam, producingL-lysine. 2. Substrate specificity:

I. may hydrolyze D-amino-lactam at a rate of 0.1 0.5 percent or so ofthe activity on L-aminolactam.

2. not hydrolyze a-butyrolactam, fi-valerolactam,

e-caprolactam, cyclic dimer, trimer and pentamer of e-aminocaproic acid,D and L-pyrrolidon carboxylic acid.

3. Active range of pH: active at pH 6 12, being optimum at about pH 9.0

4. Assay method: incubated with L-amino-lactam solution of a pH 9.0. Theamount of L-lysine produced in the incubated mixture is analyzed. 5.Optimum temperature: 70 C for an incubation of about 20 minutes. 40- 50C for an incubation of several hours.

6. Inhibitor: Ethylenediaminetetraacetic acid (EDTA) 7. Activator: Mn,Mg, Zn, Co

8. Purification: Fractionation with ammonium sulfate. Fraction of about30 40 percent saturation. Then, liquid chromatography using DEAEcellulose.

In connection with the reaction step of-this invention, more preferredembodiments are as follows:

The reaction mixture is composed of a proper amount of a L-amino-lac'tamhydrolyzing agent and an aqueous solution of L-arnino-lactam orL-aminolactam containing substance such as DL-amino-lactam, pH of 6 12,preferably 9. As for the concentration of amino-lactam in an aqueoussolution used above, 20 wt percent is preferred.

L-amino-lactam hydrolyzing agent is used preferably in an amount of 0.110 wt percent based upon dry amino-lactam. Incubation is carried out ata temperature from about room temperature to about 75 C, for the usualytime, preferably with gentle stirring.

When insolubiliized enzyme is employed, the insolubilized enzyme ispreferably used in the form of a col umn.

After completion of the reaction, the L-lysine product is recovered fromthe reaction mixture by any one of a number of different procedureswhich are conve- 'nient for such purposes, and which are well known tothose skilled in the art.

For instance, a reaction mixture is centrifuged and a supernatant liquidis obtained. The pH of the supernatant liquid is adjusted to 4.1 byadding an aqueous solution of HCl. A small amount of activated charcoalis then added to the supernatant liquid. The mixture is boiled for a fewminutes, cooled to about room temperature and filtered.

The filtrate is dried and a residue is obtianed.

The residue may be composed principally of lysine mono-hydrochlorideor amixture of lysine monohydro- 5 chloride and amino-lactam hydrochloride.

The residue is suspended in a small volume of methyl alcohol. Thesuspension is stirred for several hours at room temperature. Aminolactamhydrochloride, if present, is dissolved into methyl alcohol, but lysinel0 monohydrochloride remains. Crystals of lysine monohydrochloride areobtained from the suspension by filtration.

In order to obtain aminolactam, the following operation is employed. Thefiltrate is mixed with a dilute aqueous solution of HCl and the pH ofthe filtrate is brought down to about 1.

The solution is dried and the residue is principally composed ofaminolactam monohydrochloride and a trace amount of lysinedihydrochloride.

The residue is suspended in a small volume of ethyl. alcohol. Thesuspension is stirred for several hours at room temperature.

Lysine dihydrochloride is dissolved in ethyl alcohol, but aminolactamhydrochloride remains.

Crystals of aminolactam hydrochloride are obtained from the suspensionby filtration.

This invention is further illustrated by the following examples.

EXAMPLE 1 TABLE 4 Strains L-lysine umoles/ ml Cryplococcus laurentiiTORAY 2001, FERM-P 709 254 Cryplococcru laurentii TORAY 2002, PERM-P 710234 Trichosporon cutaneum TORAY 2004, FERM-P 712 32 Trichosporoncutaneum TORAY 2007, PERM-P H4 272 Candida humicola TORAY 2008, FERM-P715 I36 Candida hunu'cala TORAY 2010, FERM-P 717 I28 Candida humicalaTORAY 2020, PERM-P 719 264 Trichospomn cutaneum TORAY 2035, PERM-P 725I82 Candida hurnicola IFO 0735, ATCC 9949 ISI Candida humicola [F0 0760103 Trichosparon cutaneum [F0 0173 54 Trichosporan cutaneum IFO 1198 I88Cryplococcm lauremii IFO 0609 243 Cryplococcus laurenn'i IFO 0906 251EXAMPLE 2 Two hundred ml of the culture medium, the composition of whichwas heretofore described in this specification, were placed in al-L-flask and sterilized. Each yeast strain shown in Table 5 wasinoculated to the medium and cultured with aerobic shaking at 30 for 20hours. Cells were collected by centrifugation from the culture broth anddried with acetone.

The reaction mixture was composed of 10 mg/ml of the acetone-dried cellsand 5 percent DL-, L- or D- aminolactam aqueous solution adjusted withI-ICI to pH 9.

Incubation was carried out at 50 C for 2 hours.

As a result, it was found that L-aminolactam was converted to L-lysine,and D-aminolactam wa'iibt substantially hydrolyzed.

In Table 5, the concentration, expressed in [1.1110- les/ml, of lysineproduced and aminolactam remaining are shown.

TABLE D L-aminolactam L-aminolactam D-aminolactam Lysine AminolactamLysine Aminolactam Lysine Aminolactam Strains produced remainingproduced remaining produced remaining (Iryptococcus laurentii TORAY 2001131 244 140 235 0.4 390 Cryptococcus laurentii TORAY 2002.... 190 186202 177 .6 388 Trichosporon cutaneum TORAY 2004 38 350 40 344 .6 388Trichosporon cutaneum TORAY 2007... 192 185 340 35 1.8 387 Candidahumicola TORAY 2008 84 302 100 281 .4 391 Candida humicola TORAY2010.... 69 322 81 299 .4 392 Candida humicola TORAY 2020 193 187 280102 1.8 386 Trichosporon cutaneum TORAY 2035... 98 291 102 280 .4 7 390Candida humicola lFO 0735 97 290 105 277 .2 390 Candida humicola IFO0760 52 333 55 338 .3 389 Trichosporon cutaneum IFO 0173.. 23 361 29 360.7 387 Trichosporon cutaneum IFO 1198.. 110 275 l 280 .7 387Cryptococcus laurentii IFO 0609. 191 188 385 0 .4 390 Cryptococcuslaurentii lFO 0906. 190 175 387 O .4 390 Not inoculated 0 390 0 390 390EXAMPLE 3 Cryptococcus laurentii TORAY 2001 FERM-P 709 was cultured inthe medium the composition of which was heretofore described in thisspecification at 30 C for hours. Cells were harvested and dried withacetone.

The reaction mixture was composed of 1 g of the acetone dried cells and100 ml of a 10 percent DL- aminolactam aqueous solution adjusted withHCl to pH 9.

Incubation was carried out at 50 C for 20 hours with gentle stirring.

From the reaction mixture 6.4 g of L-lysine monohydrochloride and 5.4 gof D-aminolactam hydrochloride were obtained.

The yield of L-lysine obtained from DL-aminolactam was 45 percent. Theoptical purity of the L-lysine obtained was 100 percent.

The yield of D-aminolactam from DL-aminolactam was 42 percent. Theoptical purity of the D- aminolactam was 98.5 percent.

EXAMPLE 4 Acetone-dried cells of Cryptococcus Iaurentii TORAY 2002,PERM-P 710 were prepared in the same manner as in Example 3.

The following operations were carried out in much the same way as inExample 3, except that the acetonedried cells prepared here were used.

From the reaction mixture 6.7 g of L-lysine monohydrochloride (yield: 47percent, optical purity: 98.2 percent) and 5.1 g of D-aminolactamhydrochloride (yield: 40 percent, optical purity: 98.2 percent) wereobtained.

EXAMPLE 5 Acetone-dried cells of Candida humicola TORAY 2020, FERM-P 719were prepared in the same manner as in Example 3.

The reaction mixture was composed of 1g of the acetone-dried cells and200 ml of a 5 percent DL- percent) and 5.3 g of D-aminolactamhydrochloride (yield: 41 percent, optical purity: 97.8 percent) wereobtained.

EXAMPLE 6 Cryptococcus laurentii TORAY 2001, FERM-P 709 was cultured ina medium the composition of which was heretofore described in thisspecification at 30 C for 20 hours. The cells were harvested andlyophilized.

The reaction mixture was composed of l g of the lyophilized cells and mlof a 10 percent DL- aminolactam aqueous solution adjusted with HCl to pH9. Incubation was carried out at 50 C for 20 hours with gentle stirring.

From the reaction mixture 6.0 g of L-lysine monohydrochloride (yield: 42percent, optical purity 99.4 percent)and 5.8 g of D-aminolactamhydrochloride (yield: 45 percent, optical purity: 99.0 percent) wereobtained.

EXAMPLE 7 Cryptocaccus laurentii TORAY 2002, FERM-P was cultured in amedium the composition of which was heretofore described in thisspecification at 30 C for 20 hours. The cells were harvested andsuspended in water.

The reaction mixture was composed of 50 ml of the intact cell suspension(1 g of dry weight) and 50 m1 of 20 percent DL-aminolactam aqueoussolution adjusted with HCl to pH 9. Incubation was carried out at 40 Cfor 20 hours.

From the reaction mixture 4.6 g of L-lysine monohydrochloride (yield: 32percent, optical purity: 99.2 percent) and 5.8 g of D-aminolactamhydrochloride (yield: 45 percent, optical purity 97.6 percent) wereobtained.

EXAMPLE 8 Cryptococcus laurentii TORAY 2001, FERM-P 709 was cultured ina medium the composition of which was heretofore described in thisspecification at 30 C of 20 percent DL-aminolactam aqueous solution and30 ml of water. The pH of the reaction mixture was adjusted to 9 withHCl.

Incubation was carried out at 40 C for 30 hours. From the reactionmixture 6.1 g of L-lysine monohydrochloride (yield: 43 percent, opticalpurity: 99.0 percent) and 5.3 g of D-aminolactam hydrochloride (yield:41 percent, optical purity: 100 percent) were obtained.

EXAMPLE 9 Fifty ml of cell extract which was dialyzed against 10*Mphosphate buffer was prepared from 25 g of living cells of Cryptococcuslaurentii TORAY 2001, FERM-P 709 in the same manner as in Example 8.

The cell extract was fractionated with ammonium" sulfate'and a fractionof 30 40 percent saturation was obtained. This fraction was dialyzedagainst 10*"M phosphate buffer and lyophilized and 21 mg of crude enzymewas obtained. The L-aminolactam hydrolyzing activity of this crudeenzyme was 700 umoles/hr/mg.

The reaction mixture was composed of 10 mg of the crude enzyme and 50 mlof 10 percent DL- aminolactam aqueous solution adjusted with HCl to pH9.

Incubation was carried out at 43 C for hours.

From the reaction mixture 3.0 g of L-lysine monohydrochloride (yield: 43percent, optical purity 99.6 percent) and 5.9 g of D-aminolactamhydrochloride (yield 46 percent, optical purity 99 percent) wereobtained.

EXAMPLE l0 Ten mg of the crude enzyme prepared in Example 9 wasdissolved in 5 ml of 5 X 10"M phosphate buffer (pH 7.6). One g ofDEAE-cellulose (OH type) was bufferized with the same buffer. Bothpreparations were mixed, stirred for 30 minutes, and filtered. Thus, anenzyme adsorbed on DEAE-cellulose was obtained.

A column, 1 cm (1) X 20 cm, was made of the enzymeadsorbingDEAE-cellulose.

Six hundred ml of 1 percent DL-aminolactam aqueous solution (the pH wasnot adjusted) was allowed to flow through the column which was kept at43 C for 20 hours.

From the effluent 4.0 g of L-lysine monohydrochloride (yield: 47percent, optical purity percent) and 3.8 g of D-aminolactamhydrochloride (yield: 49 percent, optical purity 98.5 percent) wereobtained.

I claim:

l. A process for preparing L-lysine which comprises contacting ana-amino-e-caprolactam selected from the group consisting of L- andDL-a-amino-ecaprolactam in an aqueous solution with a microorganismhaving L-aminolactam-hydrolyzing activity selected from the groupconsisting of the genera Cryptococcus, Candida and Trichosporon whereinsaid aminocaprolactam is subjectedto asymmetrical hydrol ysis by thecatalytic action of said microorganism.

2. A process for preparing L-lysine which comprises contacting ana-amino-e-caprolactam selected from the group consisting of L- andDL-a-amino-ecaprolactam in an aqueous solution with the enzyme,L-aminolactam amido-hydrolase isolated from cells of a microorganismhaving L-aminolactam-hydrolyzing activity selected from the generaCryptococcus, Candida, and Trichosporon, said aminocaprolactamundergoing asymmetrical hydrolysis in contact with said enzyme.

3. A process for preparing L-lysine which comprises subjectinga-amino-e-caprolactam in an aqueous solution to the action of a memberhaving L-aminolactamhydrolyzing activity selected from the groupconsisting of growing culture, a culture broth, living cells, driedcells, and a cell extract of a microorganism selected from the generaCryptococcus, Candida and Trichosporon, said aminocaprolactam undergoingasymmetrical hydrolysis in contact with said group member.

4. The process according to claim 1 wherein L-lysine so produced in thereaction mixture is then isolated in a conventional manner.

2. A process for preparing L-lysine which comprises contacting an Alpha-amino- epsilon -caprolactam selected from the group consisting of L-and DL- Alpha -amino- epsilon -caprolactam in an aqueous solution withthe enzyme, L-aminolactam amido-hydrolase isolated from cells of amicroorganism having L-aminolactam-hydrolyzing activity selected fromthe genera Cryptococcus, Candida, and Trichosporon, saidaminocaprolactam undergoing asymmetrical hydrolysis in contact with saidenzyme.
 3. A process for preparing L-lysine which comprises subjectingAlpha -amino- epsilon -caprolactam in an aqueous solution to the actionof a member having L-aminolactam-hydrolyzing activity selected from thegroup consisting of growing culture, a culture broth, living cells,dried cells, and a cell extract of a microorganism selected from thegenera Cryptococcus, Candida and Trichosporon, said aminocaprolactamundergoing asymmetrical hydrolysis in contact with said group member. 4.The process according to claim 1 wherein L-lysine so produced in thereaction mixture is then isolated in a conventional manner.